1. Field of the Invention
This invention relates to a special use of a live vaccine, to new live vaccines not having been used before, to a method of producing such vaccines as well as to suitable vaccine strains, especially salmonella.
2. Description of the Related Art
The majority of salmonella-conditioned Gastroenteritis infectiosa of humans are caused by contaminated animal products. Especially chicken and chicken eggs, respectively, being infected with the at present predominantly occurring serovar Salmonella enteritidis have increasingly been causing infections, recently. Nevertheless, generally all food stuffs are affected which originate from animals kept in mass-rearing. Here, normally many animals are kept in confined space, promoting the spread of infections among the animal stock.
The risk of a transmission of the infection from the infected animal to humans can be reduced by customary veterinary medical measures for the interruption of infection chains. Furthermore, thorough compliance with I kitchen hygiene regulations during the processing of contaminated animal products can prevent a transmission to humans. However, especially the latter regulations are not always being considered during the storage and processing of food. Therefore, it is imperative to rule out the possibility of infected animals being processed right from the beginning. This can be achieved e.g. through a vaccination of the animal stock against salmonella infections.
Suitable salmonella live vaccines have to comply with various different conditions:
1. The virulence of the vaccine strains used in the production of the vaccines has to be adjusted in a way that guarantees a non-apparent infection on the one hand, and a sufficient persistence of the vaccine strains in the host tissue on the other hand, as a prerequisite for high immunogenicity.
2. Furthermore, the stability of the vaccine strains used with respect to their virulence and their protective properties has to be widely assured, I.e. it has to be assured that they do not mutate back into the virulent wild strain.
3. To allow for the reduction of the probability of infections it should be ascertained that the vaccine strains are not permanently being excreted alive and that they can only service for a short period of time in the environment, respectively.
The above-mentioned three conditions, which a live vaccine has to comply with, are to be discussed in detail in the following. As described in 1, the production of a suitable salmonella live vaccine is based on a reduction of the virulence (attenuation) of the pathogenic salmonella and simultaneous preservation of their antigen structures, and thus, the immunogenic effect in the host. One possibility is e.g., to employ deletion mutants, e. g. pur or aro auxotrophic clones, as vaccine strains. The attenuation level of these vaccine strains depends upon the lack of metabolites in vivo, which possibly impedes an accurate adaptation to the host to be immunized. In this respect, it is referred to the EP 0 263 528 in which stable asp mutants of Salmonella typhimurium with different virulence reduction levels are described. Vaccine strains with attenuation levels adapted to each of the different host species can be produced by selecting suitable asp mutants.
A further possibility for an attenuation consists of the employment of vaccine strains, the virulence reduction of which can be traced back to a metabolism drift mutation (called stwd mutation or marker in the following). The term xe2x80x9cmetabolism driftxe2x80x9d comprises all essential enzymes and functionally important cell compartments, respectively, having been functionally altered by mutations, as e.g. ribosome proteins, gyrase, RNA polymerase, permease, wherein, as a result of these mutations, translation, DNA replication, DNA transcription or permeation are more or less distinctly disturbed. Such stwd mutants, furthermore, show a resistance with respect to specific antibiotics and other substances (noxious substances). Stwd mutants can easily be obtained in laboratories as chromosomal antibiotic resistance mutants. In this respect, from the EP 0 263 528 e.g. stwd mutants with a resistance against nalidixic acid (Nal), streptomycin (Sm) or rifampicin (Rif) are known. Especially if several stwd markers are incorporated into one vaccine strain (double or triple marker vaccine strain), de facto unlimited possibilities are obtained for the production of a desired attenuation level adapted to suit every specific host species.
With respect to the prior art xe2x80x9cattenuation by means of stwd mutationsxe2x80x9d it is referred to the following publications: DD-WP 155 294; DD-WP 218 834; DD-WP 235 828; DD-WP 253 182; DD-NP 253 184; DD-WP 281 118; DD-WP 294 420; EP 0 263 528.
A further (mentioned above under 2) condition is that the attenuated vaccine strains obtained by mutation do not mutate back into the virulent wild strain. The required stability can, on the one hand, be achieved by only employing vaccine strains with which no reversions can be detected in vitro or whose reversion ratios are  less than 107. A further possibility is to employ vaccine strains comprising several mutations which independently reduce virulence. Here, the probability of a back mutation can almost be excluded.
The final condition, mentioned above under 3 in connection with the term xe2x80x9cinterruption of infection chainsxe2x80x9d, especially concerns the risks with respect to a possible excretion and permanent survival of the vaccine strains outside the vaccinated host. In this respect, it is desirable to reduce the excretion and the capability of survival of the vaccine strains in the environment. To guarantee a sufficient immune response, on the other hand, the capability of temporary survival of the vaccine strains in the host tissue after e. g. oral or parenteral application should only be slightly impaired or not at all. Vaccine strain mutants complying with such requirements are known e.g. from the DD-WP 218 836, DD-WP 231 491, DDWP 253 182, DD-WP 253 183, DD-WP 253 184, and EP 0 263 528. In the prior art it is suggested to optimize suitable vaccine strains by employing so-called anti-epidemic markers for the reduction of excretion and the capability of survival in the environment. The term anti-epidemic marker characterizes outer envelope mutations in a broader sense, causing a functional variation of the permeability barrier in the outer membrane.
Vaccine strains can be provided with different anti-epidemic markers depending upon the intended application form. The anti-epidemic markers known at present are divided into three groups, depending on the alterations they cause in the outer membrane of the vaccine strain. The first group comprises the so-called hst markers. The incorporation of an hst marker causes the vaccine strain to become highly sensitive towards bile, anionic detergents, macrolide antibiotics and other noxious substances. Owing to the high sensitivity towards bile, there is a reduced excretion with feces caused by the inactivation of the vaccine strains already occurring in the intestinal lumen. If vaccine strain bacteria are excreted, they only have a shortened survival time in the environment, due to the lack of the permeability barrier in the outer membrane against tensides and macrolides and other noxious substances. Therefore, an infection can almost be excluded when using vaccine strains including hst markers. When employing the usual doses of vaccine, vaccine strains comprising hst markers can only be applied parenterally, however. If applied orally, due to the high sensitivity towards bile, the virulence is influenced to such an extent that a sufficient immune response can only be achieved by employing extremely high doses of vaccine. Therefore, the solution for an oral application would be to provide vaccine strains including an anti-epidemic marker from one of the other two known groups. One group comprises the so-called rbt markers (reversion to bile tolerance). The rbt marker can be obtained by mutation from the hst marker. It provides the vaccine strain with an anti-epidemic potency just as the hst marker does. However, in contrast to the hst marker the vaccine strain comprising an rbt marker is tolerant towards bile, and can therefore be applied orally without a reduction of the virulence impairing the vaccination effect. The same stands for a further group, the so-called rtt marker (reversion to tenside tolerance). The rtt marker can be obtained by mutation from the. rbt marker. The vaccine strain comprising the rtt marker is tolerant towards tensides and simultaneously possesses a sufficient anti-epidemic potency due to the remaining high sensitivity towards macrolides and other noxious substances. Also the rtt marker strain can be applied orally without any problems.
Taking this information and these publications as a basis, live vaccines can be produced which comply with all conditions required. The adaptation to the respective host can be effected, e.g. in animal test series.
A further problem remains to be solved. Even the compliance with all precautions does not exclude the possibility of a person dealing e.g. with the vaccination of the animals or the production of the vaccines, coming into contact with the in fact attenuated but, nevertheless, still pathogenic salmonella vaccine strains. Where healthy people are concerned, there is hardly any risk of an infection. However, if the immune system of people is weakened (e.g. by an HIV-infection), the contact with such vaccine strains can result in a salmonella infection.
Therefore, the object of the invention is to provide a live vaccine against salmonella infections, starting from the known prior art, being optimally attenuated for the host to be immunized, providing it with an immunity for reducing the excretion of wild strains when applied orally or parenterally, and constituting only a minor risk for people, especially those with a weakened immune system, or none at all. A further object of the invention is to provide a method for producing salmonella live vaccines optimally suited to the respective host by employing significantly less animal experiments than the conventional methods. Finally, the invention is to provide salmonella live vaccine strains for live vaccines suitable for chickens and poultry in general.
This object is attained by a specific live vaccine, a specific method for the production of salmonella live vaccines, and live vaccine strains.
The art described in this section is not intended to constitute an admission that any patent, publication or other information referred to herein is xe2x80x9cprior artxe2x80x9d with respect to this invention, unless specifically designated as such. In addition, this section should not be construed to mean that a search has been made or that no other pertinent information as defined in 37 C.F.R. xc2xa71.56(a) exists.
The invention concerns the use of a live vaccine strain known as such (see e.g., EP 0 263 528) for the production of a specific live vaccine. The known salmonella live vaccine strains comprise an envelope marker as well as an attenuation marker (e.g. auxotrophy marker or stwd marker) providing them with an anti-epidemic potency (reduced excretion by the host and reduced survival rate in the environment, respectively). The envelope marker being employed there, furthermore, causes a sensitization of the vaccine strains towards macrolide antibiotics. This antibiotic sensitization has, hitherto, merely been employed for the selection of suitable envelope mutants, i.e. in the production of vaccine strains. According to the invention it has been recognized for the first time that the macrolide sensitivity of the vaccine strains comprising an envelope marker can also function as a safety mechanism when employing the vaccine strains produced in this manner. The vaccine is to be designed such that, in the case of it causing an infection of another host, an effective therapeutical treatment of the infected host can be carried out by means of macrolides. This aim is achieved relatively easily by selecting only such vaccine strains (provided with an envelope marker) for the production of the vaccine whose propagation can be controlled by the application of justifiable doses of macrolide antibiotics.
The known salmonella live vaccine strains used in a specific manner show a sensitivity towards macrolides, due to their envelope marker. In addition to envelope mutants having an increased sensitivity towards hydrophobic antibiotics (macrolides, e. g. erythromycin) also other envelope mutants are described (I. A. Hancock, R. E. W.: Ann. Rev. Microbial. 1984, 38, 237-264), occasionally having different permeabilities with respect to a sensitivity towards hydrophilic, hydrophobic and polycationic antibiotics. Such envelope markers have been insignificant in the production of bacterial live vaccines, hitherto.
The invention also concerns live vaccines which are produced by including at least one attenuated live vaccine strain having an envelope marker providing the vaccine strain with an increased sensitivity towards a specific therapeutically effective antibiotic with the exception of macrolides. The live vaccine strains being employed in the production of the salmonella live vaccine according to the invention, therefore, comprise an envelope marker generally providing them with an anti-epidemic potency (interruption of infection chains) and, optionally, a sensitivity towards macrolides, but, in any case, an increased sensitivity towards another specific therapeutically effective antibiotic. The vaccine strains can be detected relatively easily by employing the selected specific therapeutically effective antibiotic, so that the production of a vaccine strain provided with a suitable envelope marker does not create a great problem. It is understood that with respect to an optionally required possibility of treatment for an unwanted infection caused by the vaccine, preferably such antibiotics are selected which constitute good results with respect to the salmonella serovars employed.
Furthermore, it is understood that the selected live vaccine strains are derived from predominant serovars, those of salmonella chosen for practical application including Salmonella typhimurium and Salmonella enteritidis, accession numbers DSM 9361 and DSM 8432.
As mentioned above, there are various possibilities for the attenuation of live vaccines. One possibility is to include an auxotrophy marker (e.g. aspxcx9c) into the vaccine strain. Preferably, the vaccine strain is provided with at least one chromosomal antibiotic resistance mutation. The term chromosomal antibiotic resistance substantially comprises the above-mentioned stwd markers. It has been proven that by employing selected stwd markers and by a specific combination of several such markers, respectively, the attenuation level of the vaccine strain can be adapted optimally. Upon the selection of such chromosomal antibiotic resistance mutations for the attenuation of vaccine strains, it has to be secured that they do not exclude the increased sensitivity caused by the envelope marker towards the therapeutically effective antibiotic. Therefore, during development of the vaccine strain it is sensible to firstly determine which therapeutically effective antibiotic is to be employed for the possibility of treatment of the vaccine strain. Dependent upon this, the vaccine strain and its chromosomal antibiotic resistance mutation, respectively, are selected for the attenuation of the vaccine strain.
According to a further preferred embodiment of the invention, the envelope marker is selected such that it provides the vaccine strain (besides an anti-epidemic potency and an optional macrolide sensitivity) with an increased sensitivity towards an antibiotic of the group including quinolons, chloramphenicols or tetracyclines. Especially preferred is an envelope marker providing the vaccine strain with an increased sensitivity towards the antibiotic ciprofloxacin, presently the most effective antibiotic against salmonella. During the production of the latter vaccine strain, it is especially advantageous to provide a metabolism drift mutation, resulting in a streptomycin and/or rifampicin resistance, for the attenuation. These antibiotic resistances do not interfere with a possible sensitivity of the vaccine strain towards the antibiotic ciprofloxacin.
Depending on the desired effective spectrum, the salmonella live vaccine according to the invention can be produced from one or several vaccine strains of different serovars of the O-groups B (e.g. Salmonella typhimurium), D (e. g., Salmonella enteritidis), C (e.g., Salmonella infantis) and E (e.g., Salmonella anatum) as mono, bi, tri or tetra-vaccine.
A further problem, already mentioned above, is constituted in that, due to different sensitivities, a specifically adapted live vaccine has to be provided for every host. In this respect, it is referred to the already traded Salmonella typhimurium vaccine xe2x80x9cZoosaloral Dessauxe2x80x9d which sufficiently immunizes calves after a single oral application, however, does not protect chickens against an I. p. toxic infection until three times oral vaccination (Linde, K. et al.: Vaccine 1990, 8, 278-282). Zoosaloral is a vaccine from Impfstoffwerk Dessau-Tornau GmbH and has two auxotrophy markers Purxe2x88x92 hisxe2x88x92. The double marker attenuation is based upon the purine dependence (deficiency of purine available in the host) and an accidental co-mutation in the rfb locus adjacent to the histidin operon for the S-type polysaccharide synthesis, which co-mutation results in the leaky function. Zoosaloral is overattenuated for chicks. With respect to the sensitivity towards Salmonella typhimruium, dependent upon the host species, a hierarchy mice greater than calves greater than chickens can be constituted. From this it follows that e.g., Salmonella typhimruium vaccine strains for chicks and chickens have to have a lesser attenuation level (in comparison with mice) for the compensation of the lesser sensitivity. It can be assumed that the same correspondingly stands for other salmonella serovars.
It has been found that the generation times of salmonella live vaccines generally correlate with their attenuation levels. This correlation between generation time and attenuation level especially occurs if the vaccine strains have been provided with a chromosomal resistance mutation for the attenuation.
The relation between generation time and attenuation level permits a relatively reliable selection of vaccine strains being suitable for a specific host without prior animal experiments. In this respect, a suitable live vaccine for the immunization of chicks and chickens produced of at least one attenuated vaccine strain has a generation time thereof being about 28 to 34 minutes. Vaccine strains with such generation times have a lower attenuation level (in comparison with the vaccine strains of calves and mice), compensating the small sensitivity of chicks/chickens towards e.g., Salmonella typhimurium (S. tm) and other salmonella serovars.
As mentioned above, an effective salmonella vaccine is especially interesting with respect to the host species chicks/chickens. However, it has also been applied with ducks and ducklings and should be useful for poultry in general since they all have the relatively short lives problem that allows for the possibility of live virus in their meat. The Salmonella typhimruium stwd mutants, having been deposited/published hitherto, have no direct relation to this host species. Vaccines are introduced which can be optimally attenuated for chicks/chickens, or have already been optimally attenuated. In this respect, the vaccine strain S. tm Nal 2/Rif 9/Rtt is emphasized which is optimally attenuated for chicks/chickens. The same stands for the vaccine strain S. tm Nal 2/Rif 9 which has not been deposited, yet, with respect to this application, but still is to be commented on. The vaccine strains cited have a generation time of about 32 minutes. From this fact the xe2x80x9cattenuation equivalent generation timexe2x80x9d for the selection of optimally attenuated other strains of the same or other serovars has been derived. Nevertheless, with respect to the selection of suitable vaccine strains for chicks/ chickens generation times vary from 28 to 34 minutes. This diversity acknowledges the fact that chicks/chickens have different sensitivities towards different strains and strain-dependent differences in virulence is of each serovar, respectively. By means of a few test series, from the preselected vaccine strains having generation times between 28 and 34 minutes those can be chosen which are optimally attenuated for chicks/chickens. Advantageously, this at least allows for the animal experiments required in the preselection to be dropped.
Furthermore, with respect to the possible host chicks/chicken it is especially interesting if, as proposed according to the invention, a salmonella live vaccine is used comprising a vaccine strain with an increased sensitivity towards a specific therapeutically effective antibiotic. Chickens have a relatively short life span in comparison with other host species, as e.g., humans, calves or piglets. In general, they are slaughtered after a comparatively short rearing period and sold as frozen or fresh meat. Since the vaccination has not taken place very far in the past, it can be assumed that there are still live salmonella vaccine strain bacteria within the chickens at the time of slaughter. These can then find their way into the environment. For the normal population the small amounts of germs in question are insignificant. The risk of an infection can almost be excluded in this respect. Nevertheless, in individual cases risk patients with weakened immune systems (e.g., people with the HIV-virus) can be infected and react with clinical symptoms. Especially with respect to these people, it is required that an infection caused by the vaccine strain may be controlled rapidly and without any problems. With respect to this, the incorporation of a sensitivity towards therapeutically effective antibiotics as a safety and therapy marker is a sensible alternative, excluding all theoretical reservations.
The prototype of such a safety and therapy marker is the so-called ssq marker. Vaccine strains having an ssq marker possess a hypersensitivity towards quinolons, especially towards ciprofloxacin, at present, being the most effective antibiotic against salmonella. Also the ssq marker, like the above-mentioned hst, rbt and rtt mutations, is an envelope mutant and, therefore, also has a more or less distinctive anti-epidemic potency dependent upon its bile and anionic detergent tolerance and sensitivity, respectively.
The invention not only deals with the production of especially safe live vaccines. A further object is to provide a method for the production of a live vaccine optimally attenuated for a specific host employing hardly any animal experiments.
The principle of this method is based on the fact that an attenuation of vaccine strains (especially with respect to the incorporation of stwd markers) leads to a prolongation of the generation times in comparison with the wild strain. It has already been mentioned above that the prolonged generation times may allow for a conclusion with respect to the attenuation level of the vaccine strains. In conformity with the method according to the invention, it is therefore sufficient to determine the specific prolonged generation time of a vaccine strain suitable for a host in a single test (e.g., an animal experiment). The prolonged generation time determined such can then serve as an approximate figure for the following selection of further vaccine strains (of the same serotype), or it can be transferred to other serotypes of the same genus as an attenuation equivalent.
For the production of a vaccine optimally suited to chicks/chickens, e.g., a vaccine strain is selected, the generation time thereof being prolonged to about 28 to 34 minutes in comparison with the wild strain (22 minutes). The vaccine strain employed may e.g., be attained from a wild strain having been provided with a streptomycin (Sm) or nalidixic acid (Nal) marker. Through this, a prolongation of the generation time from 22 minutes to 25 to 29 minutes is achieved. Subsequently, a rifampicin (Rif) marker is incorporated as a further marker prolonging the generation time.
According to the essential features of the method a vaccine can be produced:
in a first step, by isolating stwd mutants of a phenotype having a specific antibiotic resistance and showing a generation time being prolonged by about 3 to 6 minutes in comparison with salmonella wild strains, for the purpose of producing strains with small or moderate attenuation,
in a second step, by again isolating stwd mutants of another phenotype with an antibiotic resistance from these lesser or moderately attenuated single marker strains, again having a generation time being prolonged by an additional 3 to 6 minutes, whereby a set of vaccine strain candidates is being achieved having prolonged generation times graded from about 28 to 34 minutes (wild strain about 22 minutes)xe2x80x94which with respect to S. tm in the mouse model (since the logarithm of an LD50 correlates linearly with the (prolonged) generation time) corresponds to graded LD50 values of about 105 to 107 (wild strain about 101) cfuxe2x80x94then once orally immunizing xe2x89xa736 hours old chicks with 109 cfu of the individual vaccine strains of this set, after two weeks orally infecting the immunized animals with 106 cfu of the wild strain, and finally favoring the S. tm Nal 2/Rif 9 strain with a (prolonged) generation time of about 32 minutes (and an i.p. LD50 mouse of about 106 cfu) as the favorite prototype vaccine strain with respect to the relation of xe2x80x9cmaximum possible prolongation of generation time/attenuation level and yet optimal reduction of the excretion of wild strainxe2x80x9d, for the determination of the xe2x80x9cattenuation equivalent (prolonged) generation timexe2x80x9d for other strains of the same serovar and serovars with only moderate or lacking virulence towards mice, respectively.
in a third step, by incorporating an xe2x80x9cssq safety and therapyxe2x80x9d marker into the double marker vaccine strains attenuated by means of stwd mutation, for the optimization of the vaccine strains/increase of the acceptance, the safety and therapy marker approximately quadrupling the sensitivity towards ciprofloxacin (chloramphenicol, doxycycline, etc.) and simultaneously insignificantly reducing the excretion and capability of survival in the environment.
The sequence described is arbitrary, and noxious substance resistance phenotypes (DD-WP 235 828) may also be used.
Furthermore, the invention relates to specific salmonella vaccine strains, having generation times of 28 to 32 minutes, as well as to variants on these vaccine strains having higher or lower attenuation levels and generation times between 28 and 34 minutes. Finally, the use of such vaccine strains for the oral immunization of chicks as well as the oral and parenteral immunization of chickens against salmonella infections is described.
The vaccine strains (with or without ssq markers) referred to in the claims and the following examples (as well as having been deposited) are examples for all the vaccine strain candidates with graded attenuation levels which may be produced. They are suitable for the production of immunogenic live vaccines, especially for chicks/chickens according to propagation methods known as such. With respect to the specifically stated vaccine strains having ssq markers, (and deposit members DSM 8433, 8435, 9362, 8434, 8441, 8432), generation times of about 32 minutes are indicated. Further specific strains having ssq markers have generation times of about 28 minutes (deposit number 9361) and about 30 minutes (deposit number 9360). This statement with respect to generation time is not to be understood as a restriction. Taking strain-dependent differences in virulence (invasive capacity/colonization activity) into account, generation times of 28 to 34 minutes are also possible as attenuation equivalents. All deposit numbers referred to herein are with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, a Budapest Treaty Depository.
The live vaccine based on Salmonella typhimruium Nal 2/Rif9/Rtt DSM 8432 has bene found to impede intestinal colonization by other bacteria or pathogens and thereby also protects the host against other infections. This effect is also predicted with the other strains of the invention.
In the following table the salmonella vaccine strains favored for the different serovars are listed.
In the following the invention is to be described in detail by several examples of embodiments.